The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurula stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus.

Amphioxus HMGB gene, AmphiHMGB, encoding a high mobility group (HMG) protein was identified from the gut cDNA library of Branchiostoma belcheri tsingtauense. Primary structural examination indicates that it is a member of HMGB family. Molecular phylogenetic analysis suggests that AmphiHMGB represents the archetype of chordate HMGB genes including HMGB1, HMGB2 and HMGB3 lthough it may have lost its C-terminal domain during evolution. Northern blotting revealed that AmphiHMGB expression was restricted mainly to guts in the adult, and AmphiHMGB transcripts were in greater abundance in gastrula and neurula though they were markedly low in 1-day larva.

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase ( proPO). The optimum temperature for activation of the proPO ranged from 30℃ to 35℃, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.

A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5±3.6, 79.17±4.5 and 13.25±4.7%, respectively. The fertilization rates of this sperm were 67.06±15.1, 76.20±10.0 and 44.93±22.6%, while the hatching rates of eggs fertilized with this sperm were 37:40 8:3, 48:18 25:7 and 23:35±0.08%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen–thawed sperm.

An oocyte-yolk protein was purified by double-step chromatography from amphioxus ovaries. The purified protein appeared to exist as a homodimer of approximately 320kDa in native polyacrylamide gel electrophoresis(PAGE), and was reduced to a single monomer of approximately 160 kDa in sodium dodecyl sulfate-PAGE(SDS-PAGE). The protein was characterized as a phospholipoglycoprotein by native PAGE and staining of gels for phosphorus with methyl green, for lipids with oil red O and Sudan black B, and for carbohydrates using periodic acid Schiff reagent. In addition, the amino acid composition of the oocyte-yolk protein was generally similar to that of vitellogenins(Vgs) isolated from different phyla of animals including both vertebrates and invertebrates. The purified phospholipoglycoprotein is thus considered as putative amphioxus Vg.